We are studying mechanisms by which mRNAs are selected for degradation by the nonsense-mediated mRNA decay (NMD) pathway. One major clue towards an understanding of this problem is the observation that a unifying characteristic of mRNAs degraded by NMD is the presence of a long 3' untranslated region (3'UTR). We have previously presented evidence that a core component of the NMD pathway, the RNA helicase Upf1, is able to act as a direct sensor of 3UTR length. Upf1 appears to accomplish this function by interacting sequence-nonspecifically with RNA, leading to preferential accumulation on long 3UTRs. We are using retroviral RNA elements that are able to inhibit decay of long 3'UTR-containing transcripts as tools to understand mechanisms of substrate selection by Upf1. These elements block the process of decay at distinct steps in the pathway, providing opportunities to probe protein and RNA requirements for both the initial steps of 3'UTR length sensing and the transition between Upf1 binding to mRNA and the initiation of decay. A 400 nt segment of the Rous sarcoma virus (RSV) gag-pol mRNA, termed the RNA stability element (RSE), has previously been shown to protect viral RNAs from Upf1-dependent decay. We have found that the RSE can inhibit decay of well characterized reporter mRNAs containing long 3'UTRs. In addition, we have identified the human PTBP1 protein as the key mediator of RSE function. Using RNAseq to characterize the gene expression profiles of cells in the presence or abscence of PTBP1 has revealed that the mechanism employed by RSV to protect its RNAs from decay is shared with as many as several hundred human mRNAs. We are currently working to understand the biochemical mechanisms and cellular consequences of this novel mode of gene expression regulation. In a separate effort, we have collaborated with James Inglese and colleagues (NCATS) to identify novel components of the human NMD pathway. High-throughput RNAi screening identified numerous candidate proteins not previously implicated in NMD. Of these, we have extensively studied the role of the ICE1 protein in linking nuclear pre-mRNA processing to the cytoplasmic decay machinery. ICE1 interacts with components of the exon junction complex (EJC), where it is important for the association between the EJC and the NMD protein UPF3B. We find that this function is important for proper regulation of many endogenous NMD targets and represents an attractive node for potential regulation and/or manipulation of the pathway. Studies of ICE1 function and additional identification of novel NMD proteins is ongoing. Finally, we have reported that a translational readthrough-promoting RNA pseudoknot from the Moloney murine leukemia virus inhibits NMD. Translational readthrough events induced by the pseudoknot appear to inhibit the process of NMD at two distinct steps. Under conditions of frequent readthrough, steady-state Upf1 accumulation in mRNPs is reduced, impairing 3'UTR length sensing. Interestingly, when the pseudoknot is used to stimulate low levels of readthrough (i.e. 1%), Upf1 accumulation in mRNPs is restored, but decay remains inhibited. We have now developed a system that allows concurrent assessment of RNA stability, translational readthrough, and Upf1 binding in order to better understand the relationship between translational readthrough and decay inhibition. We are using this system to probe the effects of readthrough stimulated via distinct mechanisms and to biochemically identify functional intermediates in the NMD pathway. These efforts include mechanistic characterization of readthrough promoted by the Moloney Murine Leukemia Virus RNase H protein.